From the work of P. J. Crosland-Taylor (Nature, vol. 171, No. 4340, pp. 37-38, Jan. 3, 1953) it becomes undoubtedly clear that he has constructed the first nozzle chamber in order to obtain a hydrodynamically focussed center-stream in combination with a sheath-stream surrounding the former stream. In his device the sheath-stream liquid is injected quasi radially through two entrance holes at the upper and thus the upstream-end of a cavity of cylindrical shape and vertical axis with a diameter larger than that of the entrance holes; this cavity is called nowadays the nozzle chamber. A suspension of blood cells is injected axially through an injection needle of 0.125 mm diameter and the sheath-stream is formed around the needle (nowadays called the nozzle) and also around the blood stream, forming in this manner the stream combination described above; the flow is directed perpendicularly downwards. With a microscope the hydrodynamically focussed blood cell suspension, i.e. the center-stream, is observed and the cells are counted optically as they scatter a light-bundle focussed upon them. After the counting process the cells and the surrounding sheath-stream leave the nozzle chamber radially through a hole located at the downstream-end of the nozzle-chamber. The center-stream (i.e. blood cells) of a diameter of about 10 micron reported by said author, was found to be wavering by about .+-.50% around its average position. It is also reported that the center-stream was found to waver even more if only one entrance hole was made for the entrance of the sheath-stream liquid. (Here a difference should be made between the sheath-stream liquid and its converted state in the nozzle chamber.) This instability can be explained in such a manner that the said sheath-stream liquid entering the chamber transfers its momentum to the liquid already present in the chamber, thereby producing a slow rotation of the said sheath-stream in the nozzle chamber approximately around the axis of the latter and as the needle (or nozzle), too, was probably not positioned exactly at the axis of the nozzle chamber, the rotating sheath-stream produced a wavering of the blood stream. The modern, so called "jet type analysers", are constructed in a manner rather similar to that of the early version of the nozzle chamber described above and the complaints about the frequent instabilities of these analysers can be partly due to the characteristic instability of the center stream as explained above in connection with the early version of the nozzle chamber.
Clearly, such wavering of said center-stream is acceptable only if the volume of analysis is large enough, which is the case with objectives of long focal length, i.e. of large focal depth. But as such objectives gather only a small fraction of the scattered or emitted fluorescent light by the cell, therefore the modified nozzle chamber, i.e. that of the jet type, came into use only after the development of high power lasers. In the jet type analyser the nozzle chamber is tapered at its down stream-end into a very thin capillary tube with an inside diameter of about 0.1 mm, and if this part of the capillary tube is UV-transparent (down to about 200 nm.) then, in principle, there are two positions where the volume of analysis can be situated. Namely, either in the center of the tube, by adjusting the focal plane of the objective there, or outside the tube where the jet-stream flows in free space in a downward direction. In the volume of analysis, the center-stream, the optical axis of the objective and the laser beam are directed quasi perpendicularly to each other in such a manner that the center-stream lies approximately in the focal plain of said objective. (See: M. A. van Dilla et al., in IEEE Transactions in Nuclear Science, NS-21, pp. 716, 1977.) If the said capillary tube is not transparent, as is the case in the droplet deflection (Fulwyler) sorter (Fulwyler, M. J., Science, vol. 150, pp. 910, 1965), then it is of course only possible to situate the volume of analysis into the open jet-stream. The instability of the stream creates instability not only in the analysis but also in the sorting operation because the perturbation in the jet-stream, caused by the instability, disturbs rather strongly the moment of droplet formation (see: Fulwyler, op. cit.) and hence, the quality of the sorting, too, which is another complaint mentioned frequently by workers in this field. An additional disadvantage of the analyser of the jet type is that it requires the use of high energy lasers. These are rather expensive and bulky, and the complete system requires an optical table in order to be mechanically stable, and its operation, because of the high-energy laser involved, is in some cases under strict safety regulations; therefore the search for an easier alternative with lower energy of illumination and with easier operating conditions is well justified, which means the reduction of the particle analyser to an equipment of the desk-set type.
It is clear that the high energy laser can be replaced by a light-source of lower intensity in the UV-region only if one can replace the objective of long focal length and of less light-gathering capability used in the jet system by another UV-objective that has a higher numerical apperture (about 1.3) and consequently has a light-gathering capability of more than one order of magnitude larger than the former objective. In such a case the cells in the center-stream should flow in a rather shallow channel of about 0.05 to 0.1 mm depth, so that the focal plane of this strong objective can be still adjusted into the center-stream, although the channel is covered by a cover glass of 0.17 mm thickness and the focal distance of the said strong objective is only about 0.24 mm.
Such a system is described by W. Dittrich and W. Gohde in the German Patent No. P 19 19 628.2, but the results of analysis were rather poor because, as follows from the description of the simple nozzle chamber (described above), the center-stream was not stable enough to make each cell flow exactly in the focal plane. Therefore, the said inventors have invented the system according to U.S. Pat. No. 3,738,759, and developed a flow-system where the cells do not flow in the focal plane of the strong UV-objective but perpendicularly to it; the cells are illuminated by a rather inexpensive and easy to operate high-pressure mercury lamp of 100 Watts, with a UV-light component of about 10 mWatts, and by using Koeler's-illumination optics in the epiillumination mode. After traversing the volume of analysis, defined earlier as the product of the sight field of the objective and the focal depth, the cells are washed away rather abruptly by a so called cleaning stream, flowing perpendicularly to the optical axis of said objective, and hence to the said particle-stream, too, into another channel which is the continuation of said channel from where the cleaning stream flows; this channel system is covered by a 0.17 mm thick cover glass and the said second channel serves also as exit-channel for the particle-electrolyte mixture which flows under the effect of a suction pump into a collecting bottle as waste. Clearly, the said channels and the vertical hole in which the cells flow in the direction of the focal plane, form a flow-system in the shape of a vertical "T". The clear advantage of this system, besides the easier illumination conditions, is that the small wavering of the center-stream does not cause great errors in the analysis of the cells, because the sight field of any objective is rather homogeneous at the vicinity of the optical axis. For this reason one would expect rather good results with this type of analyser. This is, however, not always the case because the sudden 90.degree. joining of the center and sheath-stream combination with the cleaning-stream, defined above, is prone to turbulence at the vicinity of the volume of analysis which can affect the analysis itself. That some workers still get very good results with this type of analyser is probably due to the fact that there exists a region of flow velocity for said streams at which they can join without causing an appreciable turbulence in the volume of analysis. Another disadvantage of the 90.degree. deflection of the center and sheath-stream combination is that if a small thread flows in said stream combination, this thread may not be able to follow said deflection of the streams and will get stuck at the joining volume of the channels, i.e. around the volume of analysis, causing rapid clogging in the said volume while the cells are flowing there, stopping eventually the operation of the analyser completely. That such cloggings happen rather frequently has been known from direct observations both in the said system and in other flow systems. To clean out such devices is often tedious and time consuming, not to mention the strain that ensues for the operator while working with such an unreliable analyser equipment. One more shortcoming of the flow system of W. Dittrich and W. Gohde lies in the fact that due to the turbulent flow described above and due to the bulky nature of the attached nozzle chamber, this analyser can not be readily combined with the cell sorters known to exist at the time being, which is indeed a considerable disadvantage at the time when cell sorting becomes more and more necessary.
There are two more works on cell analysers which should be mentioned for the sake of completeness. One of these is the two-parameter analyser of Severin et al. (see: E. Severin et al. in Cytometry, vol. 3, No. 4, pp. 308-310, 1983) where an argon laser beam is combined to the flow system of W. Dittrich and W. Gohde (see: op. cit.); this laser beam reaches the center-stream, and hence the cells, through a channel cut into the body of the device perpendicularly to the cleaning-exit-channel. No experimental results are given by the authors; however, as the flow system is that of W. Dittrich and W. Gohde, the general performance of this two-parameter analyser must be expected to be the same as that described above in relation with the flow system of W. Dittrich and W. Gohde. Therefore, one important disadvantage of the analyser of Severin et al. is that it can not be combined with one of the known sorter devices, either. The second analyser device is that of H. B. Steen (see: Cytometry, vol. 1, No. 1, pp. 26-31, 1980), where cells or latex beads are syringed from a jet of a center and sheath-stream combination to the top side of a microscope glass, illuminated in the epi-illumination mode, and the fluorescent light is collected by a UV-objective of relatively low magnification, located under the cover glass at that point where the jet, and hence the particles fall upon the cover glass. The results are excellent with beads of 1.5 micron and with cells of about 10 micron diameter; however, this type of device can not be combined with a sorter, either, and it is very probable that the said jet is as unstable as those of the previously described jet-analysers, because in said analyser the same type of nozzle chamber is used as in the former ones.
Presently there exists only one type of electrical analyser or sensor in practical use. In its original version, invented by W. H. Coulter, in the middle 1950s (see: van Dilla et al., op.cit.), it was used to count particles passing from a large container into another large container through an orifice with a crossection only slightly larger than that of the cell or particle to be analysed, according to its size. Both containers are filled with electrolyte; in the upstream container the electrolyte also contains the particles or cells to be analysed; in the other container are the cells which were already analysed. Electrodes connect this system to an electrical circuit and as most of the resistance of the electrical circuit is concentrated in the orifice, the passage of a cell or of a nonconducting particle through the orifice causes a sudden increase in the resistance of the liquid system, causing consequently an electrical voltage and/or current impulse in the connected electrical circuit. In a more modern version (see: U. Zimmermann et al., in Bioelectrochemistry and Bioenergetics, vol. 3, pp. 58-83, 1976) the cells are confined into a hydrodynamically focussed center-stream which is surrounded by a particle-free enveloping electrolyte, and are passing through the center of the orifice where the electrical field is to a very high degree uniform. In this manner an accurate sizing of the cells is possible because, as was found out experimentally, the amplitude of the electrical impulse is proportional to the cell (particle) volume. In both said versions, however, the cells are lost after passing through the orifice and hence they are not identifyable for further processings, such as cell sorting. In order to make such a processing possible, too, a flow-system has been developed (see: T. Zold, U. S. Pat. No. 4,237,416, 1980) where the cells flow in a closed channel system in the center-stream through a very narrow part of the channel system, used here as the orifice, and thus, they are analysed according to their volume and are capable to continue to flow in the same center-stream towards the sorter-part mentioned above (see: T. Zold, U.S. Pat. No. 4,175,662, 1972) which is also incorporated into the same device body as said analyser, in order to be sorted on the basis of the same quantity. Experiments with such systems provided excellent results even in combination with an optical sensor containing a large UV-objective, i.e. an objective of large numerical apperture and magnification, with epi-illumination using Koeler's-illumination system, in order to provide uniform illumination of the sight field of said UV-objective. A version of such a system is shown in FIG. 2 of U.S. Pat. No. 4,175,662, combined with a sorter system; the hydrodynamical focussing part of the system according to FIG. 2 is described below in greater detail because of its close relation to the system described in this specification. The hydrodynamical focussing, and hence the generation of the center-stream of said FIG. 2, called suspensionstream in the description of FIG. 2 takes place in the nozzle chamber 42, mentioned in said description as "entrance channel". The center-stream liquid flows into said nozzle chamber 42 through a very small capillary tube 44 which ends in nozzle 46. At this position the center-stream liquid comes into contact with the sheathstream liquid in container 48 and flows to the device through a flexible silicon pipe (indicated in said description and in FIG. 2 as number 42, too); the sheath-stream liquid enters the nozzle chamber 42 through a hole of a few mm length which is directed almost perpendicularly to the small tube 44 or nozzle 46; this hole is indicated only with a circle at the high stream end of the nozzle chamber 42, i.e. where the small tube 44 enters said nozzle chamber 42. Because of this geometry the small tube presents itself as an obstacle in the way of the flow of the sheath-stream liquid. Indeed the small tube splits the liquid into three parts. One takes an approximately 90.degree. turn and flows under the small tube 44 towards the tip of the nozzle 46 and the other two parts flow half-way around the said small tube and meet, with a head-on collision, at the top side of the small tube. Then these two stream-parts deflect each other by 90.degree. and both flow towards the nozzle 46 where the three stream parts join again and form the sheath-stream which envelops the center-stream emanating from the nozzle 46. This center and sheath-stream flow-combination, after passing the orifice 28, joins the two side streams flowing into the main channel through 36 and 37, as described in the text of FIG. 2. In this connection the following facts are of interest: Several flow-systems of this type were built and all were functioning in the same way. That is, when the nozzle chamber 42 was air bubble free, a very stable center-stream of high quality was obtained; by introducing hydrodynamical resistance into the flow of the center-stream liquid, center-stream diameters as small as one micron could be obtained which did not waver even when observed through a stereo microscope of a 50-fold magnification; the center-stream was dyed in these cases with black ink in order to make it distinguishable from the surrounding sheath-stream. However, an air bubble free nozzle chamber was achieved only after many startings, i.e. fill-up's, because usually an air bubble developped at that part of the nozzle chamber where the small tube 44 enters the said nozzle chamber 42 and that usually in the starting phase, i.e. when the empty channel system is being filled up with electrolyte under the suction effect of a suction pump attached through buffer bottles and silicon pipes to the exit holes of the flow-system. These air bubbles were found to be of varying size and are clearly caused by and during the head-on collision of said two stream parts of the sheath-stream liquid as the latter enters the empty nozzle chamber and as said stream parts are deflected towards the exit of said chamber, i.e. towards the orifice 28. As such air bubbles change the position and the shape of the center-stream and can even make the latter unstable, the reliability of this flow-system is very poor and this relative defficiency renders it to be only marginally useful for clinical practice where equipment of stable and high reliability is needed in order to obtain fast and reliable diagnostics.
From the described BACKGROUND ART result the following conclusions: Large nozzle chambers, especially with rotating liquid in them, are undesirable because of the wavering of the center-stream resulting from such flow-systems. Sharp (90.degree.) joinings of channels and streams must also be avoided because of the turbulence and air bubble generation in such systems, which turbulence causes a unreliability and instability of operation. Although the nozzle chamber of FIG. 2 of U.S. Pat. No. 4,175,662 is rather close to the desired solution, nevertheless its reliability should be further improved, i.e. such a new nozzle chamber should be developped where the reliability is practically 100% in order to reduce the stress that is developped in the operator by working with an equipment of relatively low reliability.
Therefore, the main object of this specification is to define a method of construction which, together with the other improved parts of the flow-system, is not only of a very high stability but also of a very high reliability, because due to the special inside wall geometry of said new chamber and channel system the formation of any air bubbles is practically impossible. This new analyser device is also improved in such aspects as sensitivity, maintenance, and operation, and extended in order to enable the use of epi- and direct-illumination of the particles flowing in the center-stream by means of lasers; these latter aspects have not been applied so far to the flow-systems similar to the one described in this specification.